This proposal aims to identify chemical inhibitors of the newly discovered Lin28 oncogenic pathway, and to establish the efficacy of these compounds as potential chemotherapeutics. let-7 microRNAs (miRNAs) function as tumor suppressors by inhibiting the expression of several oncogenes: let-7 is downregulated in numerous tumor types, low let-7 correlates with poor prognosis, and restoration let-7 expression effectively inhibits cancer growth in model systems. It was recently found that the RNA-binding proteins Lin28A and Lin28B selectively inhibit expression of let-7 and thus provides an exciting opportunity for the development of novel chemotherapeutic strategies to restore let-7 expression in cancer. Lin28A/B are normally only expressed during early embryonic development but is aberrantly reactivated in ~15% of human cancers, with a corresponding reduction in let-7 levels, and ectopic Lin28A/B expression enhances cell proliferation and promotes cellular transformation. Importantly, this effect can be abrogated when let-7 is reintroduced into the transformed cells. Conversely, depletion of Lin28A or Lin28B in human cancer cells lines results in decreased cell proliferation, migration, and tumorigenicity. Strikingly, Lin28A and Lin28B are specifically activated in the subset of tumors that are poorly differentiated and carry the worst prognosis. Lin28A recruits Zcchc11, a novel 3' terminal uridylyl transferase (TUTase) to block let-7 biogenesis. Thus Lin28A-TUTase represents an important new target for cancer treatment. Novel high-throughput screening (HTS) approaches will be developed and utilized to identify drugs that restore let-7 expression in Lin28A-expressing cancers. Two complementary assays will be utilized for HTS: a cell-based system to monitor let-7 expression, and a biochemical assay to identify inhibitors of the TUTase. Hit compounds will be validated using a series of secondary screens and the effect of these compounds on cancer cell proliferation will be examined. This methodology will translate basic science discoveries to the development of new and effective cancer treatments.